Transgenic animal

ABSTRACT

A transgenic rat containing in its genome a nucleotide sequence encoding a Ga subunit protein, which Ga protein subunit is uncoupled from regulation by Regulators of G-Protein Signaling (RGS) proteins, which Gx subunit protein is eventually the dominant-negative G188S mutant of Gax9, which nucleotide sequence is operatively associated with a neuron-specific expression control sequence, wherein the transgenic rat expresses the GA subunit protein in neural cells resulting in extended D-protein coupled receptor signaling mediated by the Ga subunit protein.

This application claims priority under 35 U.S.C. § 119 from ProvisionalApplication Nos. 60/199,209 and 60/245,473 filed Apr. 24, 2000 and Nov.3, 2000, respectively, which are each incorporated herein by referencein their entirety.

FIELD OF THE INVENTION

The invention generally relates to transgenic animals (rats and mice)and to animal models of human disease. The invention particularlyrelates to transgenic animals which can serve as animal models of humandiseases or conditions modulated by proteins that regulate G-proteinsignaling.

BACKGROUND OF THE INVENTION

The RGS (Regulators of G-protein Signaling) proteins act to desensitizeG protein mediated signal transduction by accelerating the endogenousGTPase activity of activated Gα subunits. RGS proteins have beendemonstrated to function as GAPs (GTPase accelerating proteins) for Gαo,Gαi, Gαz and Gαq subtypes of the Gα subunit (Grafstein-Dunn, et al. Mol.Brain Res. 2001, 88:113-123; for a review see Hepler, Trends inPharmaceutical Sci. 1999, 20:376-382). Therefore, RGS proteinsaccelerate the turning-off of G-protein coupled receptor GPCR signaling.RGS proteins likely modulate signal transduction of many clinicallyrelevant GPCRs within the CNS. In situ analyses by Gold et al.,(Neurosci. 1997, 17(20):8024-37) demonstrated brain specific RGS mRNAexpression. Additional in situ evaluation demonstrated overlap of mRNAexpression for RGS4, RGS7, and Gαq (Shuey et al., J. Neurochem. 1998,70:1964-1972).

The first member of the RGS protein family (SST2p) was identified inyeast using a genetically characterized mutant yeast strain (sst)supersensitive to the ligand of the pheromone response pathway (Dohlmanet a., Mol Cell Biol. 1996, (9):5194-209). The receptor for this pathwayis a G-protein coupled, seven transmembrane receptor (GPCR). Thecomponents of this yeast pathway are analogous to those in mammalianGPCR signaling. Subsequently, a dominant mutation was identified inyeast that phenotypically copied a yeast strain deleted for the yeastRGS (SST2p). The mutation also resulted in a supersensitivity to theGPCR ligand, alpha factor, which stimulated the pheromone responsepathway in yeast leading to cell cycle arrest. The dominant mutation wasidentified using genetic studies and molecular biology in yeast (Dohlmanet al., (supra), and was due to a G302S mutation in the yeast G protein(Gpa1) that rendered it insensitive to regulation by (RGS) SST2. Gpa1 ishomologous to mammalian Gαi proteins. The glycine residue is conservedin mammalian Gα subunits and is contained within the first switch regionin the Gα protein. Crystallography studies show that the switch regionsof Gαi interacts with RGS4 (Tesmer et al., Science 1997,278(5345):1907-16). The dominant phenotype (RGS insensitivity) resultingfrom the G302S mutation in the Gα protein identified in yeast can betransferred to the mammalian Gαq protein (Shuey et al., J. Neurochem.(1998, 70:1964-1972). This work extended the RGS insensitive phenotypeof the yeast protein, Gpa1, to mammalian Gαq protein (G188S). Themammalian Gα protein harboring the G to S mutation is able to bind theguanine nucleotide, but is resistant to the GAP activity of RGSproteins.

Since the identification of the first member (yeast protein, SST2) ofthe RGS protein family in 1996, the impact and biology of the RGSproteins remains to be clarified. RGS proteins are implicated to play arole in brain function, as suggested by region specific expression.

In particular, several GPCRs couple through Gαq to activate secondmessenger systems (Forse, Crit. Care Med. 2000, 32:524-30; Gudermann etal., Ann. Rev. Neurosci. 1997, 20:399-427) such as PLC, phosphotidylinositol, Diacyl glycerol, PKC and calcium. Additionally, thesemessengers can link into MAP kinase pathways to further modulatecellular responses. The Gαq coupled receptors include (but are notlimited to) the α1 adrenergic receptor, muscarinic receptors (m1, m3,m5), adrenoreceptors, N-methyl D-aspartate receptors, histaminereceptors, serotonin receptors, P2Y, and metabotropic glutamatereceptors. Many of these receptors show distinct expression patterns inthe brain. The Gαq coupled serotonin receptors (5-HT2A, 5-HT2B, 5-HT2C)are of particular interest because this neurotransmitter system istargeted by several anti-depressant therapeutics.

Of the anti-depressant therapeutics, lithium is the most commonly usedtreatment for bipolar affective disorder (Jope, Mol. Psychiatry 1999,4:21-25 and 117-28). Despite its years of usage, the therapeuticmechanism of action of lithium has not been clearly elucidated. Lithiumproduces a wide spectrum of behavioral and neurochemical effects leadingto speculation that its mechanism(s) of action relates to its effects onone or more signaling pathways: G-proteins, IP3, cAMP, wnt, β-catenin,GSK3b, etc. (Williams and Harwood, Trends Pharmaceutical Sci. 2000,21:61-64; Hedgepath et al., Basic Res. Cardiol. 1997, 92:385-90).

Despite the considerable efforts aimed at elucidating the mechanism ofaction of therapeutics such as lithium for treating bipolar disorders,there is no clear picture of how such compounds work. An understandingof how compounds such as lithium exert their effects would allow for thedesign and testing of novel therapeutics that produce a desiredtherapeutic effect while potentially avoiding adverse side effects.G-protein signaling constitutes an area where additional informationcould help elucidate specific mechanisms. Accordingly, there is a needfor model animal systems which can be used to identify mechanisms ofaction of RGS blockers and to use as a model for Gαq mediated activity,or discernment of G-protein crosstalk, in receptor function.Establishment of transgenic animals can provide insight into thebiological relevance, and potential therapeutic application, for amolecular target.

To this end, transgenic rats that express the RGS insensitive Gαq mutant(G188S) in neuronal tissue were established. These animals were assessedfor transgene expression and behaviors to implicate RGS control of Gαqmediated GPCRs of neurological importance.

SUMMARY OF THE INVENTION

The present invention provides transgenic animals having a transgenewhich functions as a dominant negative mutation, i.e., its expressioninhibits normal cellular processes. Transgenic animals of the typedescribed are useful in any situation where an unmutated endogenous generegulates or is regulated in a normal function in the animal.Introducing a dominant negative transgene makes it possible to assessgradations of activity in different animal lines, depending on the levelof expression of the transgene, as it overrides regulatory function ofthe endogenous system to varying degrees. This strategy is useful wherea knockout of the endogenous gene is fatal or where the endogenous geneis a member of a group of genes encoding similar or overlappingfunctions. In the latter case, the related genes may interact in ahierarchical or compensatory manner such that the phenotypic effect of asimple knockout fails to provide useful or accurate information of thegene's function. An example of such related genes is provided by thegenes encoding proteins of the G-protein signaling pathways whichcontrol intracellular responses to the presence of ligands likehormones, neurotransmitters, and the like outside the cell. As describedherein, the RGS proteins are components of G-protein signaling andfunction to modulate GPCR signalling upon binding ligand, such asneuronal responses to neurotransmitters in the central nervous system,in particular by terminating or attenuating the signal. The transgenicanimals exemplified in the present invention express a transgenicvariant protein which is functional in G-protein signaling, but which isinsensitive to RGS protein. In a specific embodiment, expression of amutant Gα protein uncouples it from RGS regulation. Alternatively,overexpression of Gα protein competitively inhibits RGS activity, alsouncoupling G protein signaling.

The present invention further includes a method for making a transgenicanimal that includes the steps of identifying a variant coding sequencecontrolling a dominant negative function and introducing the variantcoding sequence into animal cells to produce transgenic animal cellsthat replicate to produce a transgenic animal. The method can alsoinclude the step of providing a tissue-specific expression controlelement operationally linked to the variant coding sequence so thatexpression of the dominant negative function is confined to a desiredtissue type, such as the brain, of the transgenic animal. The method canproduce many animal lines bearing the same transgene but differing intransgene expression levels. Comparing the varied phenotypes of suchlines permits analysis of the quantitative influence of the dominantnegative transgene.

The invention also provides, as exemplified, transgenic rat linesbearing a dominant negative G protein, specifically a Gαq mutantprotein, as described in detail herein. The mutant Gαq coding sequencehas been combined with a neuron-specific promoter, Thy 1.2, a knownpan-neuronal tissue-specific promoter. Differences in expression levelamong different rat lines are attributable to different loci ofintegration within the genome. The transgene has also been combined witha readily identifiable epitope which can be used to identify andquantitate transgene expression levels by immuno-detection. Variouspsychomotor and drug response effects have been identified in thetransgenic rats, as described below. Unexpectedly, doses of a 5HT2Aagonist which were non-toxic to normal (wt) rats were lethal to thetransgenic rats of the invention. These transgenic rats also displayedincreased sensitivity to the 5HT2C induced feeding response. These andother observations demonstrated that 5HT2 mediated responses werealtered in the transgenic rats. Additionally, the transgenic rats showedincreased sensitivity to the nuscarinic agonist pilocarpine. Thetransgenic rats of the invention are useful for analyzing the spectrumof pharmacological action of psychoactive drugs and as research toolsfor determining the activities and interactions of G-proteins inregulation and modulation of nerve cell and brain function. Suchunderstanding is essential to the design and testing of pharmacologicagents for treatment of nerve and brain disorders including, withoutlimitation, depression, anxiety, obsessive-compulsive disorder,hyperactivity, sleep disorders, psychoses, schizophrenia, cognitiondisorders, pain, mood disorders, eating disorders, autism, attentiondeficit disorders, bipolar affective and the like.

DESCRIPTION OF THE FIGURES

FIG. 1 (A-C) is the nucleotide (SEQ ID No: 1) and amino acid (SEQ ID NO:2) sequence of mouse G188S Gαq.

FIG. 2 is a schematic diagram of the mutant mouse Gαq transgene.

FIG. 3 (A and B) shows global behavioral assessment in male (A) andfemale (B) Gq transgenic rats.

FIG. 4 (A and B) shows body weight effects in male (A) and female (B) Gqtransgenic rats.

FIG. 5 shows the results of the effect on body weight in LiCl diettreated rats.

FIG. 6 (A and B) shows effects on food intake in vehicle treated male(A) and female (B) Gq transgenic rats.

FIG. 7 (A and B) shows effects on food intake in 5-HT2C agonist treatedmale (A) and female (B) Gq transgenic rats.

FIG. 8 (A and B) shows effects of acute lithium on food intake invehicle and 5-HT2C agonist treated rats based on amount in grams, A) orpercent control (B).

FIG. 9 shows effects of vehicle or 5-HT2C agonist on food intake in LiCldiet treated rats.

FIG. 10 (A and B) shows prepulse inhibition and acoustic startle studiesin male (A) and female (B) Gq transgenic rats.

FIG. 11 (A and B) shows prepulse inhibition (A) and acoustic startle (B)studies in rats treated with acute lithium.

FIG. 12 (A and B) shows prepulse inhibition (A) and acoustic startle (B)studies in rats maintained on LiCl diet.

FIG. 13 (A-F) shows contextual fear conditioning and auditory cueconditioning in male (A-C) and female (D-F) Gq transgenic rats.

FIG. 14 (A-E) shows shock escape behavior in Gq transgenic rats.

FIG. 15 (A-H) shows effects on d-amphetamine on locomotor activity inmale (A-E) and female (F-H) Gq transgenic rats.

FIG. 16 (A-H) shows effects on phencyclidine on locomotor activity inmale (A-E) and female (F-H) Gq transgenic rats.

FIG. 17 shows effects of d-amphetamine on locomotor activity followingacute lithium treatment.

FIG. 18 shows effects of phencyclidine on locomotor activity followingacute lithium treatment.

FIG. 19 shows effects of d-amphetamine on locomotor activity in ratsmaintained on lithium chloride diet.

FIG. 20 shows effects of phencyclidine on locomotor activity in ratsmaintained on lithium chloride diet.

FIG. 21 (A and B) shows open field behavior (center return time) in male(A) and female (B) Gq transgenic rats.

FIG. 22 (A and B) shows open field behavior (line crossings) in Gqtransgenic rats.

FIG. 23 (A-D) shows open field behavior following acute lithiumtreatment based on number 4 occurrences in a 15 minutes session (A),center exit times (B), center Return time (C), time spent (D).

FIG. 24 (A and B) shows effects of a 5-HT2A agonist on headshakes (A)and convulsions (B) in Gq transgenic rats.

FIG. 25 shows effects of a 5-HT2A agonist on headshakes following acutelithium treatment.

FIG. 26 shows effects of a 5-HT2A agonist on convulsions following acutelithium treatment.

FIG. 27 (A and B) shows effects of a 5-HT2A agonist on headshakes (A)and lethality (B) in rats maintained on chronic lithium diet.

FIG. 28 (A-C) shows effects of a muscarinic agonist in inducing tremors(A), convulsions (B) or death (C) in Gq transgenic rats.

FIG. 29 (A-C) shows effects of a muscarinic agonist over following acutelithium treatment.

FIG. 30 (A-C) shows effects of a muscarinic agonist in inducing tremors(A), convulsions (B) and death (C) on rats maintained on a LiCl diet.

DETAILED DESCRIPTION OF THE INVENTION

The family of RGS protein contains approximately 20 members (Ross andWilkie, Ann. Rev. Biochem. 2000,69:795-827, especially 796-803).Systematic deletion of each individual RGS subtype would be an enormousundertaking. In addition, different RGS proteins are co-expressed invarious brain regions, so the effect of a deletion of a particular RGSprotein may be masked by co-expression of a different RGS. Additionally,proteins may functionally compensate for a deleted protein, a role thatmay be artificially induced by a knockout. To gain a more global insightinto the function of RGS proteins, transgenic rats were generated thatexpress a Gαq mutant that is resistant to the GAP activity of RGSproteins. Gαq is expressed at lower levels in the brain in comparison toGαo or Gαi subtypes, and therefore provides a greater opportunity toobserve a transgene effect. Toward this end, rats were generated thatexpress the mouse Thy1.2-G188S Gαq transgene (FIG. 2).

It was discovered that many of the behavioral and pharmacologicaleffects of lithium (Kofman and Petishi, Eur. Neuropsychopharmacol. 1999,9:385-97) are exhibited by the transgenic rats of the invention. Thetransgenic rats of the invention overexpress a mutant form of Gαq, whichis not regulated by RGS-protein, thus resulting in a model of RGSblockade. Similar to the Gαq mutant transgenics, wild-type rats treatedwith lithium have a mild tremor or vibration, have decreased food intakeand body weight and have deficits in prepulse-inhibition. Moreover,either acute or chronic lithium attenuates the locomotor activatingeffects of the indirect dopamine agonist amphetamine and potentiatesresponses to the 5-HT2A agonist DOI, the 5-HT2C agonist RO 60-0175, themuscarinic agonist pilocarpine, and the noncompetitive NMDA antagonistPCP. In short, the Gαq mutant animals exhibit a phenotype of lithiumtreated animals.

The parallels observed between the Gαq mutant transgenic rats andlithium treated rats indicates that Gαq mutant transgenic rats representa treated model of bipolar affective disorder. Since the Gαq mutanttransgenic animals mimic the effects of RGS-blockade, these parallelsindicate that RGS blockers are useful in the treatment of bipolaraffective disorder. Moreover, the transgenic animals represent a moreprecise model of RGS blockade than lithium treatment, and thus areuseful for studying the effects of other drugs or RGS-blocked animals.

In the disclosure below, the invention is discussed in terms oftransgenic rats. It should be apparent that the invention includestransgenic mice as shown in Example 3, below, and generally relates totransgenic rodents (e.g., rabbits, hamsters, gerbils, and guinea pigs inaddition to rats and mice), as well as other animals having a transgenewhich functions as a dominant negative. The transgenic animals of theinvention are genetically modified animals in which at least one foreigngene has been inserted into the genome. These animals allow regulatoryprocesses on the cellular level to be examined and influenced in asystematic and specific manner not achievable with any other testsystems. Transgenic animals of the type described are useful in anysituation where an unmutated endogenous gene controls an inhibitoryfunction in the animal. In particular, the transgenic animals of theinvention are useful for analyzing the spectrum of pharmacologicalaction of psychoactive drugs, and as research tools for determining theactivities and interactions of G-proteins in regulation and modulationof nerve cell and brain function, and function in other tissues. Thetransgenic animals serve as excellent models for evaluating the effectof compounds, i.e., potential or actual ligands of GPCRs, in context ofdecoupling GPCR signaling from RGS protein regulation. Suchunderstanding is essential to the design and testing of pharmacologicagents for treatment of nerve and brain disorders including, but notlimited to, depression, anxiety, obsessive-compulsive disorder,hyperactivity, sleep disorders, psychoses, schizophrenia, cognitiondisorders, pain, mood disorders, eating disorders, autism, attentiondeficits and the like.

Introduction of a dominant negative transgene makes it possible toassess gradations of activity in different animal lines, depending onthe level of expression of the transgene as it overrides the function ofthe endogenous gene, to varying degrees. The strategy is useful where aknockout of the endogenous gene may be fatal or where the endogenousgene is a member of a group of genes encoding similar or overlappingfunctions. In the latter case, the related genes may interact in ahierarchical or compensatory manner such that the phenotypic effect of asimple knockout fails to provide useful or accurate information of thegene's function. An example of such related genes is provided by thegenes encoding proteins of the G-protein signaling pathways whichcontrol intracellular responses to the presence of hormones,neurotransmitters and the like outside the cell. As described herein,the RGS proteins are a component of G-protein signaling which functionto modulate neuronal responses to neurotransmitters in the central (andperipheral) nervous system. The transgenic animals exemplified in thepresent invention possess a transgenic variant protein which isfunctional in G-protein signaling but which is insensitive to RGS.

Molecular Biology

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds.(1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins,eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];Immobilized Cells And Enzymes [IRL Press, (1986)]; B.Perbal, A PracticalGuide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

Molecular Biology—Definition

The transgenes herein may comprise a coding sequence (e.g., cDNA, asynthetic coding sequence, or genomic DNA) for a Gα subunit proteinflanked by natural regulatory (expression control) sequences, orassociated with heterologous sequences, including promoters, internalribosome entry sites (IRES) and other ribosome binding site sequences,enhancers, response elements, suppressors, signal sequences,polyadenylation sequences, introns, 5′- and 3′- non-coding regions, andthe like. The coding sequence may also be modified by many means knownin the art. Non-limiting examples of such modifications includemethylation, “caps”, substitution of one or more of the naturallyoccurring nucleotides with an analog, and internucleotide modificationssuch as, for example, those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) andwith charged linkages (e.g., phosphorothioates, phosphorodithioates,etc.). Polynucleotides may contain one or more additional covalentlylinked moieties, such as, for example, proteins (e.g., nucleases,toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators(e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactivemetals, iron, oxidative metals, etc.), and alkylators. Thepolynucleotides may be derivatized by formation of a methyl or ethylphosphotriester or an alkyl phosphoramidate linkage. Furthermore, thepolynucleotides herein may also be modified with a label capable ofproviding a detectable signal, either directly or indirectly. Exemplarylabels include radioisotopes, fluorescent molecules, biotin, and thelike.

A “coding sequence” or a sequence “encoding” an expression product, suchas a RNA, polypeptide, protein, or enzyme, is a nucleotide sequencethat, when expressed, results in the production of that RNA,polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodesan amino acid sequence for that polypeptide, protein or enzyme. A codingsequence for a protein may include a start codon (usually ATG) and astop codon.

The term “gene”, also called a “structural gene” means a DNA sequencethat codes for or corresponds to a particular sequence of amino acidswhich comprise all or part of one or more proteins or enzymes, and mayor may not include introns and regulatory DNA sequences, such aspromoter sequences, 5′-untranslated region, or 3′-untranslated regionwhich affect for example the conditions under which the gene isexpressed. Some genes, which are not structural genes, may betranscribed from DNA to RNA, but are not translated into an amino acidsequence. Other genes may function as regulators of structural genes oras regulators of DNA transcription.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease S 1), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase. The promoter may be operatively associated with otherexpression control sequences, including enhancer and repressorsequences.

Promoters which may be used to control gene expression include, but arenot limited to, cytomegalovirus (CMV) promoter (U.S. Pat. No. 5,385,839and No. 5,168,062), the SV40 early promoter region (Benoist and Chambon,Nature 1981, 290:304-310), the promoter contained in the 3′ longterminal repeat of Rous sarcoma virus (Yamamoto, et al., Cell 1980,22:787-797), the herpes thymidine kinase promoter (Wagner et al., Proc.Natl. Acad. Sci. USA 1981, 78:1441-1445), the regulatory sequences ofthe metallothionein gene (Brinster et al., Nature 1982, 296:39-42);promoter elements from yeast or other fungi such as the Gal 4 promoter,the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase)promoter, alkaline phosphatase promoter; and transcriptional controlregions that exhibit neuronal or brain specific expression, such as thegonadotropic releasing hormone gene control region which is active inthe hypothalamus (Mason et al., Science 1986, 234:1372-1378), the Thy1.2“pan-neuronal” promoter, and synapsin I promoter (Howland D, Savage M.,Huntress F., Wallace R., Schwartz D., Loh T., Melloni R., DeGennaro L.,Greenberg B., Siman R., Swanson M., and Scott R. 1995. Mutant and NativeHuman B-Amyloid Precursor Proteins in Transgenic Mouse Brain. Neurobiol.Aging. 16: 685-699), active in neurons.

A coding sequence is “under the control of” or “operatively associatedwith” transcriptional and translational control sequences in a cell whenRNA polymerase transcribes the coding sequence into mRNA, which is thentrans-RNA spliced (if it contains introns) and translated, in the caseof mRNA, into the protein encoded by the coding sequence.

The terms “express” and “expression” mean allowing or causing theinformation in a gene or DNA sequence to become manifest, for exampleproducing a protein by activating the cellular functions involved intranscription and translation of a corresponding gene or DNA sequence.Furthermore, in the context of the present invention, expressionincludes mainfestation of decoupling of GPCR signaling from RGSproteins. A DNA sequence is expressed in or by a cell to form an“expression product” such as a protein. The expression product itself,e.g. the resulting protein, may also be said to be “expressed”. Anexpression product can be characterized as intracellular, extracellularor secreted. The term “intracellular” means something that is inside acell. The term “extracellular” means something that is outside a cell. Asubstance is “secreted” by a cell if it appears in significant measureoutside the cell, from somewhere on or inside the cell.

The term “transfection” means the introduction of a foreign nucleic acidinto a cell. The term “transformation” means the introduction of a“foreign” (i.e. extrinsic or extracellular) gene, DNA or RNA sequence toan ES cell or pronucleus, so that the cell will express the introducedgene or sequence to produce a desired substance in a transgenic animal.

The terms “vector”, “cloning vector” and “expression vector” mean thevehicle by which a DNA or RNA sequence (e.g. a foreign gene) can beintroduced into a host cell, (e.g. ES cell or pronucleus) so as totransform the host and promote expression (e.g. transcription andtranslation) of the introduced sequence.

The term “heterologous” refers to a combination of elements notnaturally occurring. For example, heterologous DNA refers to DNA notnaturally located in the cell, or in a chromosomal site of the cell.Preferably, the heterologous DNA includes a gene foreign to the cell. Aheterologous expression regulatory element is such an elementoperatively associated with a different gene than the one it isoperatively associated with in nature.

The terms “mutant” and “mutation” in the context of the invention meanany detectable change in genetic material encoding a Gα subunit protein,e.g., DNA, or any process, mechanism, or result of such a change thatinvolves partial or complete uncoupling of the Gα subunit from RGSproteins. This includes gene mutations, in which the structure (e.g.,DNA sequence) of a gene is altered, any gene or DNA arising from anymutation process, and any expression product (e.g., protein or enzyme)expressed by a modified gene or DNA sequence. The term “variant” mayalso be used to indicate a modified or altered gene, DNA sequence,enzyme, cell, etc., i.e., any kind of mutant. Specific examples of suchmutations include the one corresponding to a glycine to serinesubstitution at position 188 of mouse Gαq protein, and Gαi mutationscorresponding to the mutation in yeast Gpa1 resulting in a glycine toserine substitution at position 302.

As used herein, the term “homologous” in all its grammatical forms andspelling variations refers to the relationship between proteins thatpossess a “common evolutionary origin,” including proteins fromsuperfamilies (e.g., the immunoglobulin superfamily) and homologousproteins from different species (e.g., myosin light chain, etc.) (Reecket al., Cell 50:667, 1987). Such proteins (and their encoding genes)have sequence homology, as reflected by their sequence similarity,whether in terms of percent similarity or the presence of specificresidues or motifs at conserved positions. In particular, Gα subunitproteins are homologous, particularly Gαq and Gαi families. Similarly,RGS proteins are homologous, especially with respect to the RGS domain(Ross and Wilkie, supra).

Gα Subunit Proteins and RGS Proteins

The present invention provides for expression of a mutant Gα subunitprotein, or overexpression of a normal Gα subunit protein, so thatG-protein mediated signal is uncoupled from regulation by RGS proteinswithout altering GTP binding by the Gα, or its ability to transduce asignal upon ligand binding to a GPCR. This can occur if a mutation inthe Gα protein reduces or ablates its ability to interact with one ormore RGS proteins. This can also occur on overexpression of the Gαsubunit, so that some Gα not associated with the G-protein complex bindsone or more RGS proteins, preventing them from regulating signaling bythe complex.

In specific embodiments, mutant Gα proteins contain a glycine to serinesubstitution at a position homologous to amino acid residue 188 ofmurine Gαq, or amino acid residue 302 of yeast Gpa1. Other examples ofmutants include Gαz, e.g., in which glutamine at position 205 issubstituted with leucine (Q205L).

In addition to functioning in the G-protein complex, Gα mutants of theinvention bind the RGS domain of RGS proteins with reduced affinity, ornot at all. Thus, the RGS proteins do not regulate Gα activity, i.e.,the RGS proteins do not mediate increase rate of hydrolysis of GTP boundto the Gα subunit protein. RGS proteins are described above, and in Rossand Wilkie (Annu. Rev. Biochem. 2000, 69:795-27) and Zheng (TIBS 1999,24:411-14). Particularly preferred subfamilies of RGS proteins are theRZ, R4, R7, and R12 families. Thus, mutant variants of Gα for use intransgenic animals of the invention can be readily identified byreduction or elimination of binding to one or more RGS proteins, withoutinhibiting signal function.

Transgenic Animal Preparation

Transgenic animals can be produced by several methods known to thoseskilled in the art. One method involves taking fertilized oocytes from afemale animal. A desired foreign DNA or transgene can then incorporatedinto the oocytes. Incorporation of the transgene into the oocyte can beaccomplished by several methods such as via an appropriate retroviralvector, or by microinjection.

As noted above, any transgenic animal can be used. Preferred, especiallybecause they are well studied for behavioral responses, are rats (or anyspecies, including Sprague-Dawley (Taconic Labs), Long Evans (Taconic),Fischer 344 (Taconic), and Wistar. (Taconic). Because of their closeevolutionary relationship, mice are also useful. Mouse strains includebut are not limited to BALB/C, BL-6, DBA/2, etc. In addition to Taconic,suitable rats, mice and other animals are available from Charles River,Jackson Laboratories, and colonies in accredited animal care facilities.

Techniques for creating a transgenic animal, particularly a mouse or ratare well known (Gordon, International Review of Cytology1989,115:171-229). Various approaches to introducing transgenes areavailable, including microinjection of nucleic acids into cells,developed in 1973 (in this technique, as of 1980, frequency of genetransfer ranged from 5% to 20%), retrovirus vector methods, and genetransfer into embryonic stem (ES) cells. The ES technique in particularpermitted manipulation of the cells in culture (see also Bradley and Liu(Nature Genetics 1996,14:121). For example, Capecchi developed a methodby which transgenes can be incorporated into embryonic, fetal or adultpluripotent stem cells (Science 244:1991, 1288-1292). In Capecchi'smethod, embryonic stem cells are isolated from blastocysts cultivated invitro. These embryonic stem cells can be kept stable in culture overmany cell generations without differentiation. The transgene can thenincorporated into the embryonic stem cells by electroporation or othermethods of transformation. Stem cells carrying the transgene wereselected for and injected into the inner cell mass of blastocysts. Theblastocysts were then implanted into pseudopregnant females. Since notall the cells of the inner cell mass of the blastocysts carry thetransgenes, the animals are chimeric with respect to the transgenes.Crossbreeding of the chimeric animals allows for the production ofanimals which carry the transgene. An overview of the process isprovided by Capecchi Trends in Genetics 1989, 5:70-76.

The control of gene expression is accomplished by a variety of meanswell-known in the art. Expression of a transgene can be constitutive orregulated to be inducible or repressible by known means, typically bychoosing a promoter that is responsive to a given set of conditions,e.g. presence of a given compound, or a specified substance, or changein an environmental condition such as tissue type or temperature. Inexamples described herein, the mutant Gαq coding sequence has beencombined with a neuron specific promoter, Thy 1.2, a known pan-neuronaltissue-specific promoter. The term “inducible expression” extends to anymeans for causing gene expression to take place under definedconditions, the choice of means and conditions being chosen on the basisof convenience and appropriateness for the host organism.

Transformation can be carried out by a variety of known techniques,depending on the organism, on characteristics of the organism's cellsand of its biology. Stable transformation involves DNA entry into cellsand into the cell nucleus. For organisms that can be regenerated fromsingle cells (which includes some mammals), transformation can becarried out in in vitro culture, followed by selection for transformantsand regeneration of the transformants. Methods often used fortransferring DNA or RNA into cells include micro-injection, particle gunbombardment, forming DNA or RNA complexes with cationic lipids,liposomes or other carrier materials, electroporation, and incorporatingtransforming DNA or RNA into virus vectors. Other techniques are knownin the art. For a review of the state of the art of transformation, seestandard reference works such as Methods in Enzymology, Methods in CellBiology, Molecular Biology Techniques, all published by Academic Press,Inc. N.Y. DNA transfer into the cell nucleus occurs by cellularprocesses, and can sometimes be aided by choice of an appropriatevector, by including integration site sequences which can be acted uponby an intracellular transposase or recombinase (see e.g, [Craig, Ann.Rev. Genet. 1988, 22:77; Cox. In Genetic Recombination (R. Kucherlapatiand G. R. Smith, eds.) 1988, American Society for Microbiology,Washington, D.C., pages 429-493; Hoess. In Nucleic Acid and MolecularBiology (F. Eckstein and D. M. J. Lilley eds.) Vol. 4, 1990,Springer-Verlag, Berlin, pages 99-109. Direct transformation ofmulticellular organisms can often be accomplished at an embryonic stageof the organism. For example, in Drosophila, as well as other insects,DNA can be micro-injected into the embryo at a multinucleate stage whereit can become integrated into many nuclei, some of which become thenuclei of germ line cells. By incorporating a marker as a component ofthe transforming DNA, non-chimeric progeny insects of the originaltransformant individual can be identified and maintained. Directmicroinjection of DNA into egg or embryo cells has also been employedeffectively for transforming many species. In the mouse, the existenceof pluripotent embryonic stem (ES) cells that are culturable in vitrohas been exploited to generate transformed mice. The ES cells can betransformed in culture, then micro-injected into mouse blastocysts,where they integrate into the developing embryo and ultimately generategermline chimeras (see, e.g. Haren et al, Annu. Rev. Microbiol. 199953:245-281; Reznikoff et al., Biochem. Biophys. Res. Commun. 1999,266(3):729-734; Ivics et al, Methods Cell Bid. 1999, 60:99-131; Hall etal., FEMS Microbiol. Rev. 1997, Sep:21(2):157-178; Craig Annu. Rev.Biochem 1997, 66:437-474; Beall et al. Genes Dev., 1997,11(16):2137-2151). By interbreeding heterozygous siblings, homozygousanimals carrying the desired gene can be obtained.

In general transgenic animals of the invention include any transformablespecies except humans. Of particular interest are mammals, includingknown transformable species such as mouse, rat, sheep, and pig, andothers, as transformation methods are developed, including bovine andnon-humans primates and model organisms such as C. elegans, zebra fish,and yeast (S. cerevisiae, S. pombe and P. Pastoris).

Phenotype

As used herein, the term “phenotype” includes biochemicalcharacteristics, such as extended GPCR signaling, i.e., due touncoupling of RGS protein regulation of GPRC signaling, to physicalmanifestations of such uncoupling in the animal like increasedsensitivity to various compounds and GPCR, and behavior changes in theanimal. As used herein, the term “uncoupling” means inhibition orblockade of RGS protein interaction with the Gα protein, whether byaction of a Gα mutant or by overexpression of Gα.

A variety of behavioral tests are known to those skilled in the artwhich can be used to characterize transgenic animals and which can beused to determine the therapeutic indications of transgenic animals andanimals used in the methods of the invention. Such behavioral testsinclude, but are not limited to, open field, global assessments, bodyweight, food intake, startle and prepulse inhibition, contextual fearand auditory cue conditioning, responsiveness to shock, pharmacologicalresponses, and locomoter activity.

Uses of Transgenic Animals of the Invention

The transgenic animals of the invention can be used to identifycompounds or compositions that block RGS function or activate or prolongGα signaling. This is defined herein as “modulating activity”.Particularly, the transgenic animals of the invention are useful foranalyzing the spectrum of pharmacological action of psychoactive drugs,and as research tools for determining the activities and interactions ofG-proteins in regulation and modulation of nerve cell and brainfunction. Such understanding is essential to the design and testing ofpharmacologic agents for treatment of nerve and brain disordersincluding, but not limited to, depression, anxiety, obsessive-compulsivedisorder, hyperactivity, sleep disorders, psychoses, schizophrenia,cognition disorders, pain, mood disorders, eating disorders, autism,attention deficits disorders, bipolar affective disorders, and the like.

Agents That Mimic The Phenotype of the RGS Insensitive Transgenic Rats

The results presented in this application support the parallels observedbetween the RGS insensitive Gαq mutant transgenic rats, and lithiumtreatment of wildtype rats; thereby indicating that an agent (e.g.,small molecules, peptides, antibodies, antisense oligonucleotides)capable of modulating RGS function (i.e., blocking) can be used to treatdiseases treated by lithium, such as bipolar affective disorder.

Comparative use of the animals described herein can be used to identifynovel therapeutics. For example, unknown agents (compounds or peptidesetc.) can be administered (using various concentrations) to untreatednormal rats, lithium exposed normal rats, and untreated transgenic rats.Animals are scored for agent effect in a plurity of tests similar to(but not limited to) behavioral analysis as described herein.

Key tests for the lithium phenotype are those that specifically relateto the IP3 pathway, including RO food intake, DOI headshakes, andpilocarpine cholinergic signs, all of which link to Gq-mediate receptorsignals. These are the preferred minimum tests for establishing a linkto the effect of lithium.

In one embodiment, untreated control rats are administered a testcompound, or vehicle, while transgenic rats receive vehicle only.Comparison of these three groups (wildtype+vehicle, wildtype+compound,transgenic rat+vehicle) using one or more behavorial phenotype or otherphenotype characterization of tests such as those described herein,allows for scoring differences in behavior. Wildtype animals thatreceived a potential agent that scored similarly to transgenicrats+vehicle, identify an agent which functions through a mechanismsimilar to that observed in the transgenic rat. If the results of thesetests in the transgenic animals are substantially the same as thoseobtained in the treated group then the drug interacts as in the RGSpathway and can be used to treat, e.g., bipolar diosorders. As usedherein, the term “substantially the same” means that the samequalitative (alteration of phenotype) or quantitative (shift in potency)change. For example, transgenic rats and lithium treated rats both haveuncreased sensitivity to DOI (a qualitative change), and similar shiftsin potency of RO and pilocarpine (a quantitative change).

EXAMPLES

The following examples are provided as illustrations of specificpreferred embodiments and are not intended to limit the scope of theinvention.

Example 1 Cloning of The TransgenePCR Amplification and Cloning of TheGαq G188S Transgene

Plasmid pcDNAamp Gqsst (DiBello et al. J. Biol. Chem. 1998,273:5780-5784) was used as a template to PCR a 1.1 kb fragment of themouse Gαq cDNA. Gqsst contains a G to A conversion at nucleotide 573 anda G to C conversion at nucleotide 575 that converts a glycine residue toserine at amino acid residue 188 (G188S) (see FIG. 1). In addition,several nucleotide changes were incorporated 5′ to the mutation toconvert an endogenous Gαq amino acid sequence portion to that of an EEepitope tag (FIG. 1) to allow mutant Gαq protein detection using anti-EEantisera. “EE” refers to a two glutamate sequence that is part of anepitope recognized by a commercially available mouse monoclonal antibodyGlu-Glu (Babco, Berkley Antibody Company, Richmond, Calif.). Thisantibody was raised against the sequence CEEEEYMPE and is specific foreither six amino acid sequences EYMPME or EFMPME. The EE epitope isknown to be insertable into many G proteins without altering function.Primers (summarized in table 1) DH1, DH2, DH3, and DH4 were used inoverlap PCR with elongase polymerase (BRL) to PCR the 1.1 kb mutant GαqDNA. Primer DH1 contains an Xho I restriction endonuclease sequence justupstream of a consensus Kozak sequence (CCACCATG) which was incorporatedinto the 5′ end of the Gαq cDNA. Primer DH4 overlaps the TAA stop codonalso contained the Xho 1 restriction site enabling the 1.1 kb PCRproduct to be cloned as an Xho I restriction fragment. In the firstround of PCR, primers DH1 and DH2 were used to generate a 0.9 kb DNAfragment in which nucleotide T at position 926 bp was converted to a Cto destroy an endogenous Xho I restriction site. Primers DH3 and DH4were used to generate the 3′ end 0.2 kb of Gαq DNA which had partialoverlap with the DH1/DH2 product at the destroyed Xho I site. The secondstep of PCR was done using products DH1/DH2 and DH3/DH4 and outsideprimers DH1 and DH4 to generate the final 1.1 kb mutant Gαq cDNA flankedby Xho I restriction sites. The 1.1 kb Xho I fragment waselectrophoresed using a 1% agarose gel and was eluted by Gene-Clean(Bio-101) and subcloned into vector pGemT (Promega, Madison, Wis.). DNAsequence analysis confirmed that the 1.1 kb Gαq cDNA was authentic andcontained the expected nucleotide changes for the glycine at position188 change to serine (G188S) mutation as well as the incorporated EEepitope tag and modified Kozak consensus sequence.

TABLE 1 Seq Id PCR Primer Sequences Coordinates No. DH1GTTAAGCTTCTCGAGCCACCATGACTCTGGAGTCCATC  +1 to +29 3 bp Gαq DH2ATTCTCGGGCTGCCTGGGCATCTCTCTGG +933 to +905 4 bp Gαq DH3CCCAGGCAGCCCGAGAATTCATCCTGAAAATG +916 to +947 5 bp Gαq DH4GGCGATCCCTCGAGTTAGACCAGATTGTACTC +1097 to +1074 6 bp Gαq DH10GAGCTTGTGGAATGATCC +410 to +427 7 bp Gαq DH14 ACCATTGTGCATGAGCC +349 to+332 8 BP Gαq DH15 CCTACATCGACCATTCTG +631 to +614 9 bp Gαq DH16GGATCTCAAGCCCTCAAG +2502 to +2520 10 bp Thy1.2

Example 2 Construction of Tissue Specific Transgene Expression ConstructCloning of Thy1.2-Gαg (G188S)

Plasmid Gαq (G188S)-GemT was cut with Xho I and the 1.1 kb Gαq cDNA wasexcised from a 1% agarose gel and eluted by Gene-Clean (Bio-101)according to manufacturer's protocol. Plasmid Thy1.2 containing a 6.7 kbNotI-PvuI mouse Thy1.2 minigene cloned in pUC19 was describedpreviously. The 1.1 kb Gαq DNA was ligated to Thy1.2 vector DNA cut withXho I and treated with calf intestinal alkaline phosphatase to remove 5′phosphate ends (Sambrook et al., 1982). Clone Thy1.2-Gαq (G188S)containing the 1.1 kb mutant Gαq cDNA in the correct orientation in theXhoI cloning site of the Thy1.2 expression cassette was verified byrestriction mapping as well as DNA sequence analysis (see FIG. 2).

Example 3 Preparation of Transgenic Rat Transgene Preparation For DNAMicroinjection

Construct Thy1.2-Gαq G188S DNA (100 micrograms) was cut with NotI andPvuI restriction enzymes overnight at 37° C. The DNA was electrophoresedon 1% low melting point agarose gel (BRL) to allow separation of the 7.8kb Thy1.2-Gαq G188S transgene DNA from smaller sized 2 kb and 1 kbfragments generated from restriction of the pUC19 vector (New EnglandBiolabs) backbone. The 7.8 kb fragment was excised from the LMP gel andheated to 70° C. to melt the agarose. Once liquefied, an equal volume ofphenol was used to extract the gel mix. The aqueous portion wasre-extracted using a phenol-chloroform mix followed by chloroform. Theaqueous fraction was then precipitated using 2 volumes of 95% ethanol,followed by a 70% ethanol wash. The DNA pellet was resuspended in 2.5 mlTE buffer. One gram of cesium chloride (Sigma, St. Louis, Mo.) was addedfor every 1 ml of DNA-TE mix and gently dissolved. Ethidium bromide (10mg/ml) was added to a final concentration of 0.74 mg/ml and the samplewas loaded into 3.9 ml heat sealable ultracentrifuge tubes (Beckman) andspun at 100,000 rpm for 4 h at 25° C. in a Beckman Optimax table topultracentrifuge. Linear Thy1.2-Gαq G188S was eluted from the CsClgradient and extracted 5 times with isopropanol to remove the ethidiumbromide. The DNA was then dialyzed against microinjection buffer (5 mMTris-HCl, pH 7.4, 0.1 mM Na₂ EDTA) overnight. Dialyzed DNA was dilutedto 2 mg/ml for microinjection.

Transgenic Rat Production

Transgenic rats were produced using modifications of proceduresdescribed by (Hogan et al., Manipulating the Mouse Embryo. Cold SpringHarbor, N.Y.: Cold Spring Harbor Laboratory Press; 1986). Glass needlesfor micro-injection were prepared using a micropipet puller andmicroforge. Injections were performed using a Nikon microscope withHoffman Modulation Contrast optics, with Narashigi micromanipulators anda pico-injector driven by N₂ (Narashigi).

Fertilized Sprague-Dawley rat embryos were surgically removed from theoviducts of superovulated female rats and placed into M2 medium. Cumuluscells were removed from the embryos with hyaluronidase at 300 μg/ml. Theembryos were then rinsed in fresh M2 medium, and transferred into M16medium for storage at 37° C. prior to injection. After injecting the DNAsolution into the male pronucleus, embryos were implanted intoavertin-anesthetized SD recipient females made pseudo-pregnant by matingwith vasectomized males. Embryos were allowed to develop to term, andthe newborn rats were analyzed for the presence of the transgene asdescribed below.

Founder Identification

A transgene-specific polymerase chain reaction (PCR) was developed toscreen for founder transgenic rats. To obtain samples for PCR analysis,rat tail biopsies were at 6 to 8 days postnatal, and the biopsy materialdigested in proteinase K buffer containing 50 mM Tris-HCl, pH 7.5, 100mM Na₂EDTA, 100 mM NaCl, 1% SDS, 1.6 mg/ml proteinase K at 56° C.overnight. Extracts were then spun at 14,000 rpm for 15 min to removedebris. To prepare for PCR, proteinase K digested samples were diluted1:200 in water and then heated for 15 min at 95° C. 1 μl of diluted heatinactivated sample was then used in the PCR reaction. The DH16 primersequence lies approximately 100 bp upstream of the ATG translation startcodon in exon 2 of the Thy1.2-Gαq transgene. Primer DH14 lies 318 bp 3′to the ATG start in the Gαq coding sequence. PCR amplification was doneusing PCR supermix (Life Technologies, Gaithersburg, Me.) 22 mMTris-HCl, pH 8.4, 55 mM KCl, 1.65 mM MgCl₂, 220 μM each nucleotide(dATP, dCTP, dTTP, dGTP), 22 units recombinant Taq DNA polymerase, and 1μl DNA sample. PCR was performed using the following step conditions:94° C., 5 min, 1 cycle, followed by 94° C., 1 min, 60° C., 1 min, 72°C., 1 min for 30 cycles, followed by 72°then a 4° C. soak using a MJResearch PTC 200 thermocycler. The 420 bp PCR product was resolved on a2% Nusieve agarose-1% Seakem agarose gel (FMC).

A total of 11 founder rats were identified using the transgene specificPCR. Of these 10 rats were confirmed to contain integrated transgene DNAby Southern blot analysis (Sambrook et al., 1978). Founder number 80 waslater shown to be nontransgenic. Two founders (# 37, # 64) died prior toreaching sexual maturity however the remaining 8 founders all producedtransgenic f1 progeny upon mating to wild types.

Example 4 Preparation of Transgenic Mice Transgene Preparation and DNAMicroinjection

Construct Thy1.2-Gαq G188S DNA (100 micrograms) is obtained and treatedas described above in Example 3. Because this is a murine sequence it isused identically as described in Example 3 above for production oftransgenic rats to produce transgenic mice.

Transgenic mice are produced using modifications of procedures describedby (Hogan et al., Manipulating the Mouse Embryo. Cold Spring Harbor,N.Y.: Cold Spring Harbor Laboratory Press; 1986). Glass needles formicro-injection are prepared using a micropipet puller and microforge.Injections are performed using a Nikon microscope with HoffmanModulation Contrast optics, with Narashigi micromanipulators and apico-injector driven by N₂ (Narashigi).

Fertilized mouse embryos are surgically removed from the oviducts ofsuperovulated female mice and placed into M2 medium. Cumulus cells areremoved from the embryos with hyaluronidase at 300 μg/ml. The embryosare then rinsed in fresh M2 medium, and transferred into M16 medium forstorage at 37° C. prior to injection. After injecting the DNA solutioninto the male pronucleus, embryos are implanted intoavertin-anesthetized recipient females made pseudo-pregnant by matingwith vasectomized males. Embryos are allowed to develop to term, and thenewborn mice are analyzed for the presence of the transgene as describedbelow.

Example 5 Analysis of Transgene Expression Expression Analysis ofThy1.2-Gαq Transgenic Lines

To determine the levels and sites of expression of the mutant Gαqtransgene in the rat, an RNase protection assay capable of detectingboth transgene-derived Gαq and endogenous rat Gαq was done on brain andperipheral tissues including liver, kidney, spleen and heart for each ofthe Thy1.2-Gαq transgenic lines. A 221 bp fragment of mouse Gαq cDNA wasamplified by PCR using primers DH10 and DH15 that encompasses the Gαq EEepitope tag as well as the G188S mutation. The 221 bp product by clonedinto vector pGemT (Promega) to yield plasmid 1015/GemT and was verifiedby DNA sequence analysis. Plasmid 1015/GemT was linearized with NcoI onthe 5′ end of the insert in the polylinker of the pGemT vector and wasused as a template for riboprobe synthesis. The riboprobes weresynthesized using an in vitro transcription kit (Promega) and theprotocols as described by the vendor. The 319 base riboprobe (221 basesGαq plus 98 bases polylinker sequence) was synthesized in 1×transcription buffer containing 10 mM DTT, 0.05 mM each GTP, ATP, CTP,10 μM ³²P-labeled UTP (Amersham; 600 Ci/mmol activity), 1 μg Nco Ilinearized 1015/GemT and 5 units of SP6 RNA polymerase for 1 h at 37° C.followed by treatment with RQ1 DNAse for 30 min at 37° C. Labeled RNAwas separated over a G50 spin column (Boehringer Mannheim, Indianapolis,Ind.) and the eluate was counted by liquid scintillation.

The RNAse protection assay was done as previously described (Howland etal., Neurobiol. Aging. 1995, 16: 685-699) using RNAse A/T1 mix. TotalRNA was isolated using Trizol (Life Technologies) from whole brain orheart, liver, kidney, thymus as previously described (Howland et al.supra. The products of the RPA were electrophoresed on a 5%polyacrylamide/8M urea gel followed by exposure overnight to X-ray film(Kodak). Quantitation of transgene Gαq and endogenous Gαq bands was doneusing a Storm phosphoimager (Molecular Dynamics). For comparison,transgenic rats harboring the mutant Gαq cDNA driven by theneuron-specific rat synapsin I promoter (Howland et al., supra) was alsoanalyzed in the RPA assay.

Using RNAse protection, the expected 221 base protected productrepresenting the transgene-derived Gαq mRNA was apparent in allThy1.2-Gαq transgenic brain samples. Smaller molecular weight productsof 80 to 120 bases represent protection of the endogenous mouse GαqmRNA. For comparison, transgenic rats harboring the mutant Gαq cDNAdriven by the neuron-specific rat synapsin I promoter (Howland et al.,supra was also analyzed in the RPA assay. In each case, expression ofthe mutant Gαq RNA driven from the mouse Thy1.2 promoter was more robustthan that seen from any of the Syn-Gαq transgenic lines. The 221 baseprotected Gαq transgene RNA product was not apparent in any surveyedtissue outside of the brain confirming that expression of the Thy1.2minigene cassette is restricted to the CNS and PNS (Caroni, P. 1997.Overexpression of growth-associated proteins in the neurons of adulttransgenic mice. J. Neurosci. Methods, 71 (1): 3-9). Quantitation of Gαqtransgene expression in brain indicates that lines 67, 76 and 77 expresshigher levels than endogenous rat Gαq mRNA in brain. Lines 58 and 75express comparable levels, while lines 11 and 70 express lower levels oftransgene Gαq mRNA relative to endogenous Gαq in the brain.

Immunoblot of Gαq Protein Expression

Rats were euthanized with CO₂ and decapitated. The brains were thenremoved and frozen directly on dry ice. For Western blot analysis,one-half brain per animal was homogenized in 5 ml 1×RIPA solution (50 mMTris-HCl, 150 mM NaCl, 2 mM Na₂EDTA, 1% Triton X-100(x-octylphenoxypolyethoxyethanol), 0.25% sodium deoxycholate, 0.1% SDS,1 mM benzamidine (Sigma), 0.05 mM leupeptin (Sigma), 0.02 mM pepstatin A(Sigma). Extracts were centrifuged at 14,0000×g, and supernatants wereremoved and assayed for total protein concentration by the Bradfordassay (Bradford, Anal. Biochem. 1976, 72: 248-254). Fifty mg totalprotein was electrophoresed on 8% SDS-polyacrylamide gels and blotted tonitrocellulose (Howland et al., supra). Filters were blocked overnightin TBS containing 5% non fat milk and then treated with either 1:1000rabbit anti-Gαq/G11 antibody or 1:500 mouse anti-EE antibody (BerldeyAntibody Co.) for 4 h at room temperature. Following serial washes withTBS containing 0.05% Tween 20 (polyoxyethylene sorbitan monolaurate,blots were incubated with either goat anti-rabbit or goat anti-mouse IgGconjugated to horseradish peroxidase (BioRad; 1:2000) for 1 h. Followingserial washes with TBS/0.05% Tween 20, filters were treated with ECLreagent (Amersham) and exposed to X-ray film to visualize the bands.

Detection of the protein product of the transgene was determined usinganti-EE antisera to detect a strongly immunoreactive signal from each ofthe transgenic brain samples. A polyclonal antibody which recognizesboth Gαq and G11 by Western blots was used to also detect mutant Gαqexpression in the Thy1.2-Gαq transgenic rat brains. Brain extracts fromthe Thy1.2-Gαq transgenics show overexpression of Gαq protein whencompared to nontransgenic (SD) brain samples. Highest levels of mutantGαq protein were detected in brains from Thy1.2-Gαq line 5, 67 and 76,which is in accordance with the high mRNA levels as determined by RNaseprotection assay. These data indicate that Gαq driven from the Thy1.2promoter is capable of overexpressing normal endogenous levels of Gαq inbrain.

In situ Analysis

To localize transgene Gαq RNA expression in the subregions of the ratbrain, in situ hybridization analysis was done using Thy1.2-Gαq G188Slines 11, 58, 67, 75 and 76. Animals were euthanized by CO₂ followed bydecapitation and the brains were removed and frozen on dry ice andstored at −70° C. Brain sections were cut in the coronal plane on aBright model OTF cryostat. In situ hybridization was performedessentially as previously described (Lewis et al., Biochem. Biophys.Res. Comm. 1993, 196:553-60) Mounted sections were treated withparaformaldehyde for 1 h at 25° C. Sections were washed 5 times in 2×SSCfollowed by incubation in 0.5 μg per ml proteinase K at 37° C. for 1 h.After a 1 min wash in water, sections were acetylated with 0.25% aceticanhydride in 0.1 M triethanolamine for 10 min and then washed with waterfor 5 min. Sections were then dehydrated through graded alcohols. The1015/GemT riboprobe, as described above, was labeled using ³³P-UTP(>1000 Ci/mmol, NEN) and 15,000,000 cpms were added per slide andhybridized in 50% formamide hybridization buffer at 52° C. overnight.The following day, coverslips were removed in 2×SSC and washed 5 times.Slides were incubated in RNAse A (Ambion) for 30 min at 37° C. and thenrinsed in 2×SSC. Following washes in 1×, 0.5×, and 0.1×SSC at roomtemperature, slides were washed in 0.1×SSC at 70° C. for 1 h. Slideswere then washed in 0.1×SSC at room temperature for 5 min followed by awater wash for 5 min. After dehydration through graded alcohols. Slideswere air dried and exposed to X-ray film (Kodak) overnight.

To further localize transgene Gαq mRNA expression within subregions ofthe rat brain, in situ hybridization analysis was performed usingThy1.2-Gαq G188S rat lines. These lines (11, 58, 67, 75 and 76)demonstrated a range of mRNA expression levels in brain as shown byRNAse protection analysis. It has been previously documented (Caroni, 71(1): 3-9) that the integration site of the Thy1.2 transgene can affectthe pattern of transgene expression in the brain. These reported dataindicate that certain brain regions (i.e., hippocampus, cortex, andamygdala) consistently exhibit Thy1.2-transgene expression across alllines. However, other regions including the striatum and hypothalamusexhibit variability in expression across transgenic lines carrying theThy1.2 expression cassette. In situ hybridization studies with theThy1.2-Gαq transgenic brains has confirmed the findings of Caroni (1997)in terms of expression pattern differences. Thy1.2-Gαq lines 11 and 76,demonstrated similar expression patterns. These two lines exhibited themost pan-neuronal expression patterns of all the lines analyzed. Inparticular, transgene expression in these lines was evident in thehypothalamus as well as the striatum. Expression patterns were similarbetween lines 58 and 67. These two lines show an absence of transgeneexpression in the hypothalamus and striatum. Outside of these two areashowever, distribution of transgene mRNA was similar to that observed forlines 11 and 76. Thy1.2-Gαq line 75 exhibited an expression pattern thatwas more intermediate to lines 11, 76 and lines 58, 67. Only very lowlevels of transgene mRNA were detectable in the striatum andhypothalamus of this line. A summary of the expression profile of mutantGαq in Thy1.2-Gαq lines 11, 58, 67, 75, and 76 is shown in Table 2.

TABLE 2 Gαq RNA Localization in Thy1.2-Gaq Transgenic Rat Brains 58 6711 75 76 Cerebral cortex ++ ++++ +++ +++ ++++ Thalamus + ++++ +++ +++++++ Caudoputamen − − ++ + + Accumbens nu − − ++ + + Amygdala ++ +++ ++++ ++++ Hypothalamus paraventricular nu − − +++ + ++ hypothalamic nu −− ++ + ++ (VMH, VMHDM, VMHC) arcuate nu − − + + + DMD − − ++ + ++Hippocampus CA1 pyramidal cells ++ ++++ ++ ++ ++++ CA2 pyramidal cells++ ++++ ++ ++ ++++ CA3 pyramidal cells ++ ++++ ++ ++ ++++ dentategyrus + +++ + + +++ Perinquductal gray ++ ++ − + +++ Red nu +++ − + + ++Pontine nu +++ +++ +++ + + Cerebellum Purkinje cells + ++ ++ + +++Granule cells + ++ ++ + +++ Deep cerebellar +++ +++ + + +++

Example 6 Characterization of Animal Behavior Overview of BehavioralTesting Strategy

To assess the effect of the RGS insensitive G188S Gαq mutant transgeneon behavior, the following assessments were conducted in the followingorder:

Open Field Global Assessment Body Weight Feeding Studies Startle andPrepulse Inhibition* Contextual Fear/Auditory Cue ConditioningResponsiveness to Shock Locomotor Activity* 5-HT2A HeadshakesPilocarpine Cholinergic Signs** Prepulse inhibition was re-evaluated ina second cohort of Lines 58 and 75 followed by locomotor activityassessments. **Pilocarpine cholinergic signs was conducted in femalesfrom lines 67, 76, and 11 only.

Animals—General

In general, 10 male and 10 female heterozygotes of each line werecompared with a similar number of wild-type littermate controls. Testingbegan at 7-8 weeks of age. Subjects were individually housed in hangingmetal mesh cages with ad libitum access to food and water under a 12 hlight/dark cycle. For Lines 76, 67, and 11 the subjects were maintainedon powdered rodent chow for the duration of the study. Subjects fromLines 58 and 75 were adapted to powdered chow for the feeding studiesand then maintained on that chow for the remainder of the study.Subjects were evaluated in a series of tests outlined below over aperiod of approximately 12 weeks.

For acute lithium studies, male Sprague-Dawley rats (250-300 g, CharlesRiver) were used. Rats were treated acutely with LiCl (3-10 mEq/kg i.p.)or vehicle (saline) 20-24 h prior to behavioral or pharmacologicalassessment by the methods described below. Animals were group housed inthe locomotor and prepulse inhibition studies and singly housed forstudies investigating open field behavior, feeding and DOI orpilocarpine-induced behaviors.

For chronic lithium studies, male Sprague-Dawley rats (23 days of age onarrival, Charles River) were used. Following 1 week acclimation, thesesubjects were housed in groups of 5 and fed standard grain-based rodentdiet or diet containing LiCl (1.7 g/kg) beginning at 29 days of age.Food and water were available ad libitum and body weights were measuredat least twice weekly. At 45 days of age, the regular diet group wasdivided in half. One half continued on regular diet ad libitum (ad-lib),while the other half was fed a restricted amount of regular diet dailyto reduce body weights to a level equivalent to the lithium-fed group(pair-fed). Subjects were individually housed at 47 days of age.Behavioral testing began in these subjects at approximately 8 weeks ofage (after 4 weeks of lithium diet) and followed a schedule of testingsimilar to the transgenic rats (see below) while continuing feeding oneither the regular diet (ad-lib or pair-fed) or lithium diet. Behavioraltesting was interrupted after locomotor studies. At 17 weeks of age (13weeks of 1.7 g/kg Li diet), the concentration of LiCl was increased to2.2 g/kg and saline (0.9%) was provided. DOI headshake studies andpilocarpine studies were conducted between 20 and 24 weeks of age. Aseparate group of male rats (young adult 200-250 g) were exposed to LiCldiet (4.3 g/kg). Prepulse inhibition studies and locomtor activitystudies were conducted between 6 and 8 weeks of exposure.

Global Behavior Assessment

A schedule of behavioral testing was arranged to determine the phenotypeof heterozygous versus wild type littermate controls. These testsevaluated animals for a number of behaviors to assess any visual/obviouseffects due to transgene expression.

Subjects were weighed and transferred from their home cages to a rack ofmetal hanging cages in groups of 10. The rack was then moved to anobservation room, and the subjects given an acclimation period. Anobserver, blind to the genetic identity of the subject, evaluated eachsubject once every 15 min for an hour following the acclimation period.A checklist of behaviors, reflexes and autonomic signs (see Table 3 forsummary) was marked for presence of each sign. (See Crawley et al.,1998).

Only “tremors” appeared consistently and in the majority or all of thetransgenic positive rats. This behavior was described by the observer asa “vibration” upon handling and increased involuntary movement of thelower extremities when an animal was held around the torso, and thelower extremities were suspended (FIG. 3, A and B).

Observations of the rats were made after approximately 4 weeks on the1.7 g/kg lithium diet. Only “tremors” appeared consistently and in themajority of rats. The observer described this behavior as a “vibration”upon handling and restraint (Table 3).

TABLE 3 Global Assessment of Behavior: Detection of Vibration Induced byChronic Exposure to LiCl Ad Lib Pair Fed Regular Diet Regular Diet LiClDiet N 10 10 10 % Subjects Showing 0 30 90 Vibration Mean (+/− sem) 00.5 +/− 0.3 2.4 +/− 0.5 Incidence of Vibration (max score = 4)

Body Weight

Measurements were recorded across a number of time points and behavioralassays. Body weight was measured at toe clip (Day 6; lines 67, 76, and11), weaning (Day 21; lines 67 and 76), at the time of the globalevaluation(6-8 weeks; all lines), food intake studies (9-10 weeks; alllines), and prepulse inhibition studies (11-12 weeks; all lines).Repeated measures ANOVA and post hoc comparisons using least squaresdetermined statistical significance.

In general, rats expressing the transgene were observed to be somewhatsmaller than littermate controls. To assess this more accurately, bodyweight measurements were obtained at various times, as noted previously.For rat lines 58 and 75, (with the exception of line 58 males), the bodyweight difference was statistically significant (P<0.05) at the earliesttime point measured (6-7 weeks). For rat line 58 males, a significantdifference in weight was observed later, between weeks 8 and 9. For ratlines 67 and 76, transgenically negative males from both lines 67 andline 76 showed a similar growth curve. Line 67 and line 76 male +/− ratsweighed significantly less than the −/− rats, with an apparently greatereffect in line 76. Although there were no differences at day 6, by day21 there were statistically significant differences between the groups(data not shown). A similar effect occurred in line 67, 76, and 11females, with significant differences occurring by week 7-8 (FIG. 4).The plateau that occurs in all of the growth curves coincides with foodintake studies in which animals were fasted for 24 h 3 times over a 2week period and the animals were treated with a 5-HT_(2c) receptoragonist.

Rats were maintained on a lithium (LiCl) diet (1.7 g/kg) starting at 29days of age. Rats maintained on the chronic lithium diet weighed 20-30%less than those maintained on grain-based chow (FIG. 5).

Food Intake

Wild-type and transgenic rats were maintained on powdered chow in theirhome cages. Rats were fasted for 24 h prior to evaluation of foodintake. Food intake was measured at 2, 4, and 24 h timepoints followingip injection of vehicle, 1, or 1.7 mg/kg RO 60-0175. Experimentalsessions were generally conducted on Tues and Fri with 24 h fastsinitiating on Mon and Thurs. Repeated measures ANOVA and post hoccomparisons using least squares determined statistical significance.5-HT2C agonists decrease food intake via a Gαq modulated pathway.Studies using the selective 5-HT2C agonist RO 60-0175 were conducted todetermine if 5-HT2C agonist activity was modified in transgenic rats.The results are summarized in FIGS. 6 and 7. Food intake was reduced at1 or more time points following vehicle administration in male andfemale transgenic heterozygotes of all lines with the exception of line75 females, who did not show differences in food intake (2 hr timepointshown in FIG. 6; other data not shown).

To account for the baseline differences in food intake between wild-typeand transgenic rats, the effects of RO 60-0175 were calculated as apercentage of the vehicle food intake. The effects of 1.7 mg/kg RO60-0175 were greater in both males and females for transgenicheterozygotes for lines 75 and 67 (FIG. 7). The effects of 1.0 mg/kg RO60-0175 were greater in males of line 67 as well (data not shown).However, in line 75 females, the effects of 1.0 mg/kg RO 60-0175 wasgreater in wildtype than in transgenic rats (data not shown). There wasno change in the effects of RO 60-0175 in rats from lines 58, 76, or 11.These data indicate that 5-HT2C agonist effects that are mediatedthrough Gαq can be potentiated in the presence of Gαq mutants that arenot regulated by RGS proteins.

Two-hour food intake was markedly decreased following acute lithiumadministration in a separate group of rats. 5-HT2C agonists decreasefood intake via a Gαq modulated pathway. Studies using the selective5-HT2C agonist RO 60-0175 were conducted to determine if 5-HT2C agonistactivity was modified by lithium. Due to the marked decreases in foodintake produced by lithium, 5-HT2C agonist-induced decreases in foodintake are difficult to interpret. Nonetheless, as a percent of baselineintake, RO 60-0175 produced a greater decrease in food intake in lithiumtreated animals (FIG. 8).

Two-hour food intake was not affected in animals maintained on thechronic lithium diet (FIG. 9). 5-HT2C agonists decrease food intake viaa Gαq modulated pathway. Studies using the selective 5-HT2C agonist RO60-0175 were conducted to determine if 5-HT2C agonist activity wasmodified by lithium. The effects of RO 60-0175 on food intake werepotentiated in animals maintained on a lithium diet (FIG. 4).

Prepulse Inhibition and Startle

Subjects were weighed and transported from their home cages to thelaboratory testing room. Acoustic startle habituation was evaluated inthe morning, and prepulse inhibition was evaluated in the afternoon.Between testing, subjects were returned to their home cages. Foracoustic startle habituation, each subject was placed inside arestrainer mounted on a platform inside the startle test chamber (SanDiego Instruments, San Diego, Calif.). Movements of the subject weredetected by a transducer connected to a digitizer and computer.Following a 5 min habituation period at a background noise level of 64dB, a burst of white noise (120 dB, 20 ms) was delivered from a speakermounted 12 in above the rat on a random interval 15 sec schedule. Themovement of the subject in response to each startle stimulation wasdigitized over the 100 ms period following the onset of the burst andexpressed in arbitrary startle units. Data were analyzed by LSDfollowing 2-way ANOVA (transgene×trials) with one repeated measure(trials), with significance determined as p<0.05 .

For prepulse inhibition testing, subjects were again placed in therestrainer inside the startle test chamber for a 5 min acclimationperiod at a background noise level of 64 dB. After the acclimationperiod, each subject was exposed to four types of acoustic stimuli: astartle stimulus (20 ms duration, 120 dB) and three differentintensities of prepulse stimuli (20 ms duration; 69, 74 and 79 dBpresented 100 ms (onset to onset) prior to the startle stimulus). A testsession consisted of an initial startle stimulus followed by 15sequences of the four stimulus types, presented in a pseudorandom orderagainst a constant 64 dB white noise background. Intertrial intervalsaveraged 15 sec. Mean startle amplitude was determined by averaging 1001 ms readings taken from the beginning of the stimulus. PPI was definedas 100-([startle amplitude on prepulse trials/startle amplitude onstartle alone trials]×100) for each prepulse intensity. (Swerdlow etal., Ann. N.Y. Acad. Sci. 1999, 877:202-216). Data were analyzed by LSDfollowing a 3-way ANOVA (transgene×gender ×prepulse intensity) with onerepeated measure (prepulse intensity) for Lines 58 and 75, or followinga 2-way ANOVA (transgene×prepulse intensity) with one repeated measure(prepulse intensity), with significance determined at p<0.05.

Acoustic startle habituation was evaluated in rat lines, along with anassessment of sensorimotor gating (prepulse inhibition of acousticstartle). Startle habituation is influenced by changes in serotoninneurotransmission (impaired by SSRIs, and LSD; accelerated by 5-HT2Aantagonists). Prepulse inhibition (PPI) of acoustic startle response isan unlearned behavior where presentation of a weak stimulus immediatelyprior to a startle eliciting stimulus results in a dampening of themotor startle reflex response. PPI is disrupted in certain diseasestates such as schizophrenia, Huntington's disease and possibly OCD(Swerdlow et al., supra). It can be modeled in several species and isdependent on a basal ganglia circuitry and controlled by limbic corticalinputs. The 5-HT2A agonist DOI disrupts PPI and this effect is mediatedby 5-HT2a receptors in the ventral pallidum.

FIG. 10 summarizes the differences between heterozygotes vs wildtypelittermate controls of each line for prepulse inhibition. Line 58transgenic males, but not females, demonstrated a significant decrease(p<0.05) in startle habituation (data not shown). In line 67, thetransgenic females demonstrated a significant decrease (p<0.05), while adecrease trend was noted for line 67 transgenic males for startlehabituation (data not shown). No differences in startle habituation wereobserved for lines 75 ,76, or 11 (data not shown). However, all linesdemonstrated a transgenic effect on prepulse inhibition suggesting somecommonality with other phenotypes appearing in all lines (decreased bodyweight, “vibration”) (FIG. 10). Deficits in prepulse inhibition havebeen related to attentional deficits in certain neuropsychiatric diseasestates. These data reflect attentional deficits induced by the mutanttransgene expression. The lines differ in their pattern of expression ofthe transgene in basal ganglia structures. Thus, there is a reducedlikelihood that this phenotype is related to that brain region.

Acute lithium produced deficits did not affect acoustic startle. Lithiumproduced deficits in PPI at all stimulus intensities (FIG. 11).

Exposure to high concentration LiCl diet (4.3 g/kg) for one weekresulted in a decrease in prepulse inhibition (FIG. 12).

Contextual Fear and Auditory Cue Conditioning

Subjects were placed in a rectangular operant chamber and allowed tohabituate for 2 min, following which a tone was presented for 20 sec.Immediately following the tone, a 1 mA scrambled shock was administeredthrough a grid floor for 2 sec. The animals were removed from thechamber and returned to their home cages 30 sec after the termination ofthe shock. Approximately 15 h later animals were returned to theoriginal test chambers (context), and the incidence of freezing at tensec intervals was recorded for 5 min (maximum response =30). At the endof the 5 min test period animals were returned to their home cages.Approximately 60 min later, subjects were placed in a novel environment(new room, square chamber, plastic floor, red lights) and freezing wasrecorded for 5 min (novel). At the end of the 5 min period, the auditorytone was presented, and freezing behavior was recorded for 5 min (seeRudy and O'Reilly Behavior Neurosi. 1999,113:867-880; Pugh et al.,Behavioral Neurosci. 1997,111:503-11, Anagnostaras et al.,Neuropsychopharmacology 1999, 21:731-44).

To determine an effect of the RGS insensitive Gαq mutant transgene onassociative learning, contextual fear conditioning (hippocampaldependent, amygdala dependent) and auditory cue conditioning(hippocampal independent, amygdala dependent) were evaluated in ratlines. All subjects showed evidence of increased freezing in the contextand in the presence of the auditory cue compared with the novelenvironment. FIG. 13 (A-F) summarizes the differences in freezingbehavior observed in heterozygotes vs wildtype littermate controls ofeach line under each condition of the test. Line 76 females weredemonstrated significant decreases in each condition of the test (FIG.13D-F). Males of Line 76 showed only reduced contextual fearconditioning (FIG. 13A). Males of Line 67 also showed reduced contextualfear conditioning (FIG. 13A), whereas females of Line 67 showed trendsto reduced responses on all measures (FIG. 13D-F). Lines 58 and 75showed no significant effect and line 11 males showed increased freezingin all conditions (FIG. 13B). As Lines 67 and 76 show more hippocampalexpression of the transgene, these data point toward altered hippocampalfunction in these lines.

No significant effects were observed in lithium diet exposed rats (datanot shown).

Response to Shock

Since electric shock was used to examine associative learning, attemptsto evaluate the sensitivity to this stimulus using an active escapeprocedure were used. A procedure was designed where subjects were firsttrained to escape an electric shock. Subsequently, the level of shockwas reduced and incremented over trials. The percentage of trials inwhich the subject escaped the shock was plotted as a function of thecurrent of shock applied.

Animals were placed into a rectangular chamber and allowed to habituatefor 4 min. At the end of the habituation, the learning phase of theexperiment began. During both the learning and the testing phases,shocks were presented to the 1/2 of the grid floor on which the animalswas standing for a maximum of 10 sec or until the animal moved to theopposite end of the chamber (where no shock was being presented, i.e.,escaped). Percent escape behaviors and mean escape latency werecalculated across each block of 5 trials. The learning phase consistedof 5 trials at a shock intensity of 0.5 mA. Immediately following thelearning phase, a testing phase began with the presentation of 5 blocksof 5 trials at increasing shock intensities (beginning at 0.1 mA andincreasing in 0.1 mA increments) until the shock intensity reached 0.5mA. Animals were then removed from the testing chamber and returned totheir home cages. Data were analyzed using a paired t-test following2-way ANOVA (transgene by condition) with one repeated measure(condition) with significance determined at p<0.05.

FIG. 14 summarizes the differences between heterozygotes vs wildtypelittermate controls for the learning and testing phases of the assay.All lines learned to escape the shock. In the case of Line 67 there wasone subject that did not meet an acquisition criterion (greater than 50%of the trials escaped) and his data was not included in the subsequentassessment of sensitivity to shock. Males and females of Line 58 showeda greater sensitivity to shock as evidenced by a shift left of theresponse current curve (FIG. 14A). Another line showing a difference inshock sensitivity was Line 76 where females also appeared more sensitiveto shock, but males appeared less sensitive (FIG. 14A). Line 11 malesalso appeared less sensitive to shock but showed increased freezingbehavior in the contextual fear conditioning study. Since Line 58 showedno difference in contextual fear conditioning, and Line 76 males showeddecreased contextual fear conditioning, the altered sensitivity to shockappears not to play a role in the altered learning when it is observedin the mutant rats.

No consistent effects were observed in rats exposed to lithium diet(data not shown).

Spontaneous and Stimulant-Induced Locomotor Activity

To complement the observations of motor activity obtained using the openfield test, heterozygotes and wildtype littermate controls wereevaluated for spontaneous locomotor activity using an automated datacollection system and a more extended test period. Data in this test wascollected in multiple time bins over the test period allowing for someassessment of both the exploratory activity level in a novel environmentas well as the rate of habituation to that environment. Havinghabituated the subjects, a stimulant was administered to determine theresponsiveness of these subjects to the stimulant using locomotoractivity as a measure. In this study we used an indirect-acting dopamineagonist, d-amphetamine, and a NMDA excitatory amino acid antagonist,phencyclidine, to induce locomotion. The ability of d-amphetamine toincrease motor activity is dependent on dopaminergic neurotransmissionin the striatum, in particular the nucleus accumbens, but can beinfluenced by other neurotransmitters. The ability of phencyclidine toinduce locomotor activity is dependent on changes in both dopamine andglutatmate neurotransmission (Adams and Moghaddam, J. Neurosci. 1998,18:5545-54). The differences in locomotor activity observed underspontaneous and stimulant-induced conditions in heterozygotes vs.wildtype littermate controls are summarized in FIGS. 15 and 16.

Subjects were placed in activity monitors (Model RXYZCM16, Omnitech,Columbus, Ohio) equipped with infrared beams to detect movements on boththe horizontal (16×16 in field) and vertical axes. The testing room wasequipped with white noise. Following a 60 min habituation period duringwhich activity was monitored for differences in spontaneous locomotoractivity, each subject was treated with a dose of 1 mg/kg d-amphetamineip and returned to the activity monitor. Activity induced byd-amphetamine was monitored for 2 h. On a separate occasion, these samesubjects were placed in the activity monitors and allowed to habituatefor 60 min. Following this re-habituation, each subject was treated with10 mg/kg phencyclidine ip and monitored for activity for an additional 4h. The doses of d-amphetamine and phencyclidine were selected based onother studies as doses that induce a hyperactive state in normal rats(see Adams and Moghaddam, supra; Krebs-Thomson et al., Fur. J.Pharmacol. 1998, 343:135-43). Data were analyzed using LSD following a2-way ANOVA (transgene×time) with one repeated measure (time). Outcomeswere determined significant at p<0.05.

No significant change in spontaneous locomotor activity was detected inany line except in the Line 76 females, where the reduction in activityobserved in heterozygotes compared to wildtype was minimal, butconsistent over the majority of the test period. Males of Lines 58,75,67, and 11 as well as females of Line 67,76, and 11, but not males ofLine 76, showed reduced amphetamine-induced locomotor activity. Femalesof Line 58 and 75 were not tested. Because Lines 58 and 67 fail to showevidence of transgene expression in the nucleus accumbens, the reducedresponsiveness to amphetamine at the challenge dose must be based onother brain regions and/or interactions of other neurotransmitters withdopamine.

Phencyclidine locomotor activity was reduced in Line 67 males (FIG. 16).There was a slight trend toward a similar reduction inphencyclidine-induced activity in Line 76 male (p=0.07). In contrast,Line 58 males showed an increase in locomotor activity induced byphencyclidine that was delayed in the onset and resembled the pattern ofmotor activation associated with a higher dose of phencyclidine(pronounced ataxia and sedation followed by intense motor activity). Thesame delayed hyperactivity was observed in Line 76 females, Line 11males and females, and to a lesser degree in Line 67 females. Femalesfor Lines 58 and 75 were not tested. There was not a consistentphenotype associated with an expression pattern of the transgene. Since5-HT2A agonists have been reported to influence phencyclidine-inducedlocomotor activity (Krebs-Thomson et al., 1998), the current results mayrepresent an interaction of a modified serotonergic neurotransmittersystem (see results reported below with DOI head shakes).

Acute lithium decreased spontaneous locomotor activity during thehabituation period (FIGS. 17 and 18, habituation periods). Lithiumattenuated the increases in locomotor activity produced by d-amphetamine(1 mg/kg) (FIG. 17) and enhanced the locomotor activating effects of PCP(10 mg/kg) (FIG. 8).

Male rats exposed to high concentration of lithium diet (4.3 g/kg) forone week did not show any differences from control in spontaneous ord-amphetamine-stimulated locomotor activity (FIG. 19); however theserats did show enhanced phencylidine-stimulated activity (FIG. 20).

Open Field

To assess the effect on general ‘anxious’ behavior of the animal, anopen field test paradigm is used. The open field model evaluates twoprimary measures. The latency to return to the center square isindicative of an “anxiolytic-like” (decreased latency) or an“anxiogenic-like” (increased latency) effect. The number of line crossesand rears is indicative of changes in motor activity.

Typically, a rat placed within the center of a 3′×3′ square field willmove to an outside square (along a wall). Returning to the center squareis a demonstration of ‘non-anxious’ or ‘less anxious’ behavior. The timerecorded to return to the center square serves as an index to‘anxiolytic’ or ‘anxiogenic’ activity. For example, a longer return (tocentersquare) time is associated with a more anxious phenotype(anxiogenic).

Experiments were conducted in a 3′×3′ black Plexiglas open field. Theheight of the walls on the open field was 18 inches. On the floor of thefield were white lines that divided the field into 9 squares. Wild typeand transgenic rats were placed singly into the center square of theopen field. The latency of the rat to exit the center square (centerexit time) was recorded. The latency of the rat to return to the centersquare (center return time) was recorded. In addition, the number ofline crosses and the number of rears were recorded. All experiments were15 min in duration and were conducted in dimly lit rooms (2-4 lux). Wildtype and transgenic rats were approximately 7 weeks of age at the timeof these experiments. Statistical significance was determined by ANOVAand set to p<0.05.

Male transgenic heterozygote rats from line 58 and both males andfemales from line 76 showed an increased latency to return to the centerof the open field, suggesting a possible “anxiogenic-like” profile forthese animals (FIG. 21). Male transgenic heterozygote rats from lines 67and 76 showed a decreased activity profile with a decreased number ofline crosses (FIG. 22). This pattern of results across the transgenicrat lines demonstrates that these behaviors can be doubly dissociated.That is, the “anxiogenic-like” effects can occur in the presence (line76 male) or absence (line 58 male; line 76 female) of decrease in motoractivity. In addition, decreases in motor activity can occur in thepresence (line 76 male) or absence (line 67 male) of “anxiogenic-like”effects. Results of the open field test for animals subjected to theacute lithium treatment are shown in FIG. 23. Animals maintained onlithium diet did not differ from control animals (data not shown).

DOI Induced Headshakes

Wild type and transgenic rats were injected ip with either vehicle orDOI (0.001, 0.01, 0.1, 1.0 mg/kg) and were then housed singly in clearPlexiglas chambers (10″×18″×8″) for observational studies. A trainedobserver counted the number of headshakes for each rat during 2observation periods (0-30 min; 45-75 min).

Assessing the effect of the RGS insensitive Gαq mutant transgene onreceptors coupled via Gαq to the IP3 pathway in transgenic rats was aninterest. 5-HT2A agonists produce large increases in headshakes in ratsvia this pathway. DOI (−1[2,5-dimethoxy-4-iodophenyl]-2-aminopropane) isa 5-HT2A receptor agonist, which increases headshakes in rats, and ithas been used to investigate this pathway (Wettstein et al., 1999). DOIadministration was used herein to determine if 5-HT2A agonist activitywas modified in transgenic rats; results are shown in FIG. 27.

Surprisingly, transgenic rats from lines 58, 75, 67 and 76 that wereadministered 1 mg/kg of DOI were found dead within 24 hours ofadministration. Higher doses of DOI (3-10 mg/kg) were required toproduce similar effects in line 11 (data not shown). In separateexperiments, doses of DOI as high as 30 mg/kg in normal Sprague-Dawleyrats did not result in lethality. A lower dose of DOI (0.1 mg/kg)increased the number of headshakes in line 75 male transgenicheterozygote rats and showed a trend toward increased headshakes in line67 male transgenic heterozygote rats (dta not shown). These datademonstrate 5-HT2A agonist effects that are mediated through Gαq can bepotentiated in the presence of Gαq mutants that are not regulated by RGSproteins.

Studies using the selective 5-HT2A agonist DOI were conducted todetermine if 5-HT2A agonist activity was modified by acute or chroniclithium (FIGS. 25-27). Acute lithium attenuated the increases inheadshakes produced by 1 or 3 mg/kg DOI (FIG. 25). In addition, lithiumpretreatment (5.6 -10 mEq/kg) resulted in convulsions and lethality inrats treated with 8 mg/kg DOI (FIG. 26). DOI headshakes produced by 1 or3 mg/kg DOI were also reduced in rats exposed to lithium diet (2.2 g/kg)(FIG. 27). Moreover, convulsions and lethality were observed following 3mg/kg DOI.

Pilocarpine-Induced Cholinergic Signs and Convulsions

Muscarinic cholinergic agonists produce a series of cholinergic signs(chromodacryrhea, salivation, diarrhea, hypoactivity, tremors, foaming,convulsions and death) at high doses via a Gαq modulated pathway.Studies were conducted with the muscarinic agonist pilocarpine todetermine if muscarinic agonist activity was modified in the transgenicrats (FIG. 28). Studies were conducted with the muscarinic agonistpilocarpine to determine if muscarinic agonist activity was modified byacute lithium (FIG. 29 or chronic lithium (FIG. 30). Both acute andchronic lithium potentiated the cholinergic signs produced bypilocarpine.

The similarities between acute treatment with lithium (10 mEq/kg, 20-24h pretreatment), chronic lithium diet (1.7-4.3 g/kg) and Gαq mutanttransgenic rats are summarized in Table 5 below. All phenotypic effectsthat are observed in Gαq mutant transgenic rats are observed followingeither acute or chronic treatment with lithium. The phenotypic effectsobserved occur on non-pharmacologically-induced behaviors and onpharmacologically-induced behaviors. For example, lithium treated andGαq mutant transgenic rats show similar behavioral effects with respectto vibration, open field behavior, food intake, body weight and prepulseinhibition. In addition lithium treated and Gαq mutant transgenic ratsshow similar potentiations of behavioral effects induced by stimulationof Gαq coupled receptors (5-HT2C, 5-HT2A, muscarinic). Additionally,they also show similar modifications of the effects of indirect dopamineagonists (attenuate) and NMDA antagonists (enhance).

The parallels observed between the Gaq mutant transgenic rats andlithium treatment indicate that Gαq mutant transgenic rats represent amodel of lithium treated bipolar affective disorder. Since the Gaqmutant transgenic animals mimic the effects of RGS-blockade, theseparallels suggest that RGS-blockers may be useful in the treatment ofbipolar affective disorder. In addition, transgenic rats are useful forstudying the effects of compounds in RGS-blocked animals, which isimportant for evaluating drug interactions and conflicts.

TABLE 5 Comparative Profile of Acute or Chronic Lithium and Gaq mutantTransgenic Rats (a Model of RGS Blockade) Lithium (10 mEq/kg) LithiumDiet Gαq mutant 20–24 hr pretreatment (1.7 g/kg) Transgenics GlobalBehavioral Assessment — Vibration All lines vibration Open Field CenterReturn Time ↑ CRT nsd 2 of 5 lines ↑ CRT Line Crosses Marked ↓ LC nsd 2of 5 lines ↓ LC Food Intake Marked ↓ food intake nsd ↓ food intake BodyWeight — ↓ body weight ↓ body weight 5-HT2C Agonist (RO60-0175)Inhibition of Food Intake Lithium effect ↑ sensitivity to 2 of 5 linesovershadows any 2C RO 60-0175 ↑sensitivity to effect RO 60-0175 5-HT2AAgonist (DOI) ↓ headshakes ↓ headshakes 4 of 5 lines ↓ Induction of HeadShakes headshakes 5-HT2A Agonist (DOI) Flattened Body Posture (FBO),Conv and Death (8 mg/kg) Conv and Death (3 4 of 5 lines show FBP,Convulsions (CONY), Death mg/kg) cony, and death (1.0 mg/kg); higherdoses required for line 11 Muscarinic Agonist (Pilocarpine) ↑cholinergic signs ↑ cholinergic signs. 3 of 3 lines tested ↑ Inductionof cholinergic signs cholinergic signs Muscarinic Agonist (Pilocarpine)Conv and Death Conv and Death 3 of 3 lines tested conv Convulsions,Death (30 mg/kg) (10 mg/kg). and death (10–30 mg/kg) Prepulse Inhibition(PPI) ↓ PPI ↓ PPI All lines ↓ PPI Noncompetitive NMDA ↑ hyperactivity ↑hyperactivity 4 of 5 lines ↑ antagonist (PCP) hyeractivity (delayed)Locomotor Activity Indirect DA agonist (d-amph) ↓ hyperactivity nsd Alllines ↓ Locomotor Activity hyperactivity n.s.d. = no significantdifference n.t. = not tested

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

It is further to be understood that all values are approximate, and areprovided for description.

Patents, patent applications, publications, product descriptions, andprotocols are cited throughout this application, the disclosures ofwhich are incorporated herein by reference in their entireties for allpurposes.

1. A transgenic rat containing in its genome a nucleotide sequenceencoding a mutant Gαq subunit protein, wherein said mutant Gαq subunitprotein has glycine 188 substituted with serine (G188S) and wherein saidmutant Gαq subunit protein is uncoupled from regulation by Regulators ofG-protein Signaling (RGS) proteins, said nucleotide sequence beingoperatively linked with a neuron-specific expression control sequence,wherein the transgenic rat expresses the Gαq subunit protein in neuralcells resulting in extended G-protein coupled receptor signalingmediated by the Gαq subunit protein, and wherein said transgenic ratexhibits a phenotype selected from the group consisting of vibration asassessed by global behavior assessment; increased center return time asassessed by an open field test; decreased contextual fear conditioning;decreased food intake; decreased body weight; increased sensitivity to5-HT2A agonists and 5-HT2C agonists; increased cholinergic signs inresponse to muscarinic agonists; decreased prepulse inhibition;increased hyperactivity in response to N-methyl D-aspartate (NMDA)antagonists; and decreased hyperactivity in response to dopamine (DA)agonists, as compared to a wild type control.
 2. The transgenic rat ofclaim 1, wherein a G-protein coupled receptor that demonstrates extendedsignaling is selected from the group consisting of a muscarinicreceptor, a 5-hydroxytryptamine (HT)2A receptor, a 5-HT2C receptor, anN-methyl D-aspartate (NMDA) receptor, and a dopamine (DA) receptor. 3.The transgenic rat of claim 1, wherein the G188S mutation in the mutantGαq subunit protein is a dominant-negative mutation.
 4. A transgenic ratcontaining in its genome a nucleotide sequence encoding a mouse mutantGαq subunit that has glycine 188 substituted with serine (G188S),wherein said mutant Gαq subunit has a dominant-negative mutation and isuncoupled from regulation by Regulators of G-protein Signaling (RGS)proteins, said nucleotide sequence being operatively linked with aneuron-specific expression control sequence such that the mutant Gαqsubunit protein is expressed in neural cells resulting in extendedG-protein coupled receptor signaling mediated by the Gαq subunitprotein, and wherein said transgenic rat exhibits a phenotype selectedfrom the group consisting of vibration as assessed by global behaviorassessment; increased center return time as assessed by an open fieldtest; decreased contextual fear conditioning; decreased food intake;decreased body weight; increased sensitivity to 5-HT2A agonists and5-HT2C agonists; increased cholinergic signs in response to muscarinicagonists; decreased prepulse inhibition; increased hyperactivity inresponse to NMDA antagonists; and decreased hyperactivity in response toDA agonists, as compared to a wild type control.
 5. The transgenic ratof claim 4, wherein the mutant Gαq subunit does not interact with RGSproteins.
 6. The transgenic rat of claim 1, wherein the neuron-specificexpression control sequence comprises a Thy 1.2 promoter.
 7. A methodfor identifying a potential agent that modulates RGS modulation of Gαqhydrolysis of GTP, which method comprises comparing a phenotype of atest wild-type rat to which a test compound is administered to aphenotype of the transgenic rat of claim 1, wherein the test compoundmodulates RGS modulation of Gαq hydrolysis of GTP when the phenotype ofthe test rat is similar to the phenotype of the transgenic rat.
 8. Themethod of claim 7, wherein the phenotype being compared between the testwild-type rat and the transgenic rat is selected from the groupconsisting of vibration as assessed by global behavior assessment;increased center return time as assessed by an open field test;decreased contextual fear conditioning; decreased food intake; decreasedbody weight; increased sensitivity to 5-HT2A agonists and 5-HT2Cagonists; increased cholinergic signs in response to muscarmnicagonists; decreased prepulse inhibition; increased hyperactivity inresponse to NMDA antagonists; and decreased hyperactivity in response toDA agonists.
 9. The method of claim 7, wherein the G188S mutation in themutant Gαq subunit protein is a dominant-negative mutation.
 10. Themethod of claim 9, wherein the Gαq is a mouse Gαq.
 11. A method foridentifying a potential agent that modulates RGS modulation of Gαqhydrolysis of GTP, comprising comparing a phenotype of a test wild-typerat to which a test compound is administered to a phenotype of thetransgenic rat of claim 4, wherein the test compound modulates RGSmodulation of Gαq hydrolysis of GTP when the phenotype of the test ratis similar to the phenotype of the transgenic rat, and wherein thephenotype being compared between the test wild-type rat and thetransgenic rat is selected from the group consisting of vibration asassessed by global behavior assessment; increased center return time asassessed by an open field test; decreased contextual fear conditioning;decreased food intake; decreased body weight; increased sensitivity to5-HT2A agonists and 5-HT2C agonists; increased cholinergic signs inresponse to muscarmnic agonists; decreased prepulse inhibition;increased hyperactivity in response to NMDA antagonists; and decreasedhyperactivity in response to DA agonists.
 12. The method of claim 11,wherein the mutant Gαq subunit does not interact with RGS proteins. 13.The method according to claim 7, wherein the compound is a lead fortreating bipolar disorders.
 14. A method for identifying the effect of acompound on an animal in which Gαq is uncoupled from RGS proteins, whichmethod comprises evaluating the phenotype of the transgenic rat of claim1 to which a test compound is administered, wherein a change inphenotype relative to a control transgenic rat to which the compound isnot administered indicates the effect of the compound in an animal inwhich Gαq is uncoupled from RGS proteins.
 15. The method according toclaim 14, wherein the compound is a 5-HT2C agonist and the phenotype isinhibition of food intake.
 16. The method according to claim 14, whereinthe compound is a 5-HT2A agonist and the phenotype is increasedsensitivity to 5-HT2A agonists, wherein said increased sensitivity ismanifested by an effect selected from the group consisting of inductionof headshakes, flattened body posture, convulsions, and death.
 17. Themethod according to claim 14, wherein the compound is a muscarinicagonist and the phenotype is manifested by an effect selected from thegroup consisting of increase in cholinergic signs, convulsions, anddeath.
 18. The method according to claim 14, wherein the compound is anNMDA antagonist and the phenotype is an increase in hyperactivity. 19.The method according to claim 14, wherein the compound is a DA agonistand the phenotype is a decrease in hyperactivity.
 20. A method forgenerating a transgenic rat of claim 1 that has extended GPCR signaling,which method comprises introducing a nucleotide sequence encoding amutant Gαq subunit protein that has glycine 188 substituted with serine(G188S) into the genome of the rat, said nucleotide sequence beingoperatively linked with a neuron-specific expression control sequence,whereby the transgenic rat expresses the Gαq subunit protein in neuralcells resulting in extended GPCR signaling.
 21. The method of claim 20,wherein the G188S mutation in the mutant Gαq subunit protein is adominant negative mutation.
 22. The method of claim 20, wherein themutant Gαq subunit protein is mouse Gαq.
 23. The transgenic rat of claim4, wherein the neuron-specific expression control sequence comprises aThy 1.2 promoter.
 24. A method for identifying the effect of a compoundon an animal in which Gαq is uncoupled from RGS proteins, which methodcomprises evaluating the phenotype of the transgenic rat of claim 4 towhich a test compound is administered, wherein a change in phenotyperelative to a control transgenic rat to which the compound is notadministered indicates the effect of the compound in an animal in whichGαq is uncoupled from RGS proteins.